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Vetiver grass is capable of removing TNT from soil in the presence of urea
Environmental Pollution 158 (2010) 1980–1983
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Short communication
Vetiver grass is capable of removing TNT from soil in the presence of urea Padmini Das a, Rupali Datta b, Konstantinos C. Makris c, *, Dibyendu Sarkar a a
Department of Earth and Environmental Studies, Montclair State University, One Normal Avenue, Montclair, NJ 07104, USA Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA c Cyprus International Institute for Environmental and Public Health in Association with Harvard School Of Public Health, Cyprus University of Technology, Limassol, Cyprus b
Vetiver grass in the presence of urea effectively removes TNT from soil.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2009 Received in revised form 3 December 2009 Accepted 5 December 2009
The high affinity of vetiver grass for 2,4,6 trinitrotoluene (TNT) and the catalytic effectiveness of urea in enhancing plant uptake of TNT in hydroponic media we earlier demonstrated were further illustrated in this soil-pot-experiment. Complete removal of TNT in urea-treated soil was accomplished by vetiver at the low initial soil-TNT concentration (40 mg kg1), masking the effect of urea. Doubling the initial TNT concentration (80 mg kg1) significantly (p < 0.002) increased TNT removal by vetiver, in the presence of urea. Without vetiver grass, no significant (p ¼ 0.475) change in the soil-TNT concentrations was observed over a period of 48 days, suggesting that natural attenuation of soil TNT could not explain the documented TNT disappearance from soil. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: 2,4,6-trinitrotoluene (TNT) Urea Phytoremediation Chaotropic effect Bioremediation
1. Introduction The U.S. Environmental Protection Agency (U.S. EPA) has classified 2,4,6-trinitrotoluene (TNT) as a group C human carcinogen (U.S. EPA, 1991). Downward migration of TNT to groundwater from explosive-contaminated sites and related wastewater lagoons is of serious concern. Numerous military sites in the U.S. are in the process of being transferred to non-military entities under the base realignment and closure (BRAC) program. Following army base closures, military land may be offered to the public, but residual soil-TNT concentrations prohibit change of land use, unless appropriate remedial measures are taken. High costs and environmental concerns associated with most ex-situ remedial practices for TNT-contaminated soils have built interest in in-situ bioremediation practices (Makris et al., 2010). Our group has been investigating novel in-situ bioremediation methods for the restoration of TNT-contaminated sites. In a previous hydroponic study, we showed that vetiver grass exhibited high uptake capacity for soluble TNT (Makris et al., 2007a). The current study performed in a greenhouse setting showed that vetiver can remove TNT from soil as well, by utilizing the stimulative phytoremediation method (Makris et al., 2010). Stimulative phytoremediation is an in-situ bioremediation method for nitroaromatics that stems from the
synergistic combination of phytoremediation and biostimulation via the use of nutrient/chaotropic agent amendments (Makris et al., 2010). The limited phytoavailability of soil-TNT prompted us to test the stimulative phytoremediation method, using urea as a chaotropic agent, to enhance the solubility and plant uptake of TNT. Addition of urea altered the water structure, reducing the thermodynamic barrier associated with the introduction of a hydrophobic compound (TNT), thus increasing TNT solubility and plant uptake in a hydroponic setup (Makris et al., 2007a). Under conditions of similar initial TNT concentration, vetiver grass was superior to other plant species in removing TNT from aqueous media (Makris et al., 2007a,b), but its ability to take up TNT from soil is yet to be evaluated. Soil properties play an important role in controlling soil particle-bound TNT availability to plants/ trees and soil biological organisms (Pennington and Patrick, 1990). Eriksson and Skyllberg (2001) showed that mobility of TNT in soil primarily depended upon the soil organic matter (SOM) content. This short study was conducted to test the effectiveness of stimulative phytoremediation using the urea-vetiver system in enhancing TNT removal from Immokalee soil which is characterized by low SOM. The objective of this study was to evaluate the effectiveness of urea as a chaotropic agent in enhancing TNT removal by vetiver grass from TNT-contaminated soil. 2. Methods
* Corresponding author. Tel.: þ357 25002398. E-mail address: [email protected] (K.C. Makris). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.12.011
The soil-pot study was conducted in a greenhouse setting with the following treatments: i) three TNT concentrations (0, 40, 80 mg kg1); and ii) two chaotropic
P. Das et al. / Environmental Pollution 158 (2010) 1980–1983 agent (urea) concentrations (0 and 1000 mg urea kg1). Assuming that TNT would be less available for plant uptake from soil when compared to the hydroponic system (Thompson et al., 1998), 1000 mg urea kg1 (1045 kg urea-N ha1assuming a 15 cm soil depth) rate was used, which was the highest urea concentration tested during the hydroponic study (Makris et al., 2007b,c). This is also the highest concentration of applied urea that complies with current agronomic and environmental guidelines (Xiao et al., 2004). The Immokalee soil (pH 6, >90% sand, and 0.8% soil organic matter) (Sarkar et al., 2005) was collected from the surface horizon in the Southwest Florida Research and Education Center, Immokalee, Florida. Vetiver plants were allowed to acclimatize for 2 weeks in uncontaminated (no TNT) Immokalee soil. After two weeks, plants were transferred to the TNT-spiked soil pots, reaching a uniform plant density of 30 0.5 g kg1. Three TNT-free control soil pots were set up with vetiver grass to compare the potential toxic effects of TNT on TNT-amended plants. Six plant- and urea-free, TNTamended soil pots (40 and 80 mg kg1TNT) were also included to investigate any TNT losses due to biodegradation. All treatments were performed in triplicates. Pots were wrapped with aluminum foil to prevent potential TNT photodegradation. Experiments were carried out until near complete removal of TNT (12 days) from the spiked soil with 40 mg kg1 TNT. Soil samples were collected after 3 days to monitor soil TNT removal kinetics by vetiver grass. Soil microbial community can play major role in decreasing soil TNT by transforming TNT to metabolites (Hughes et al., 1997). The biological augmentation of TNT in soil was investigated by including plant-free, TNT-amended soil pots in the greenhouse for 48 days; soil samples were collected after 0, 12, 22, 32, 41, and 48 days for TNT estimation. Residual TNT in soil was extracted using the USEPA 8330 method, and analyzed using HPLC system (Prostar, Varian inc., USA) equipped with a UV/VIS absorbance detector (U.S. EPA, 1997; Makris et al., 2007b). Reaction rates of TNT removal by vetiver grass from soil were calculated as described by Pavlostathis et al. (1998), and Makris et al. (2007b). Statistical analyses were performed using the JMP IN version 5.1 (Sall et al., 2005).
3. Results and discussion After 12 days of exposure to soil TNT, vetiver plants did not show any phytotoxic symptoms for the 40 mg kg1 TNT load. For the 80 mg kg1 TNT load, vetiver developed yellow coloration on leaves after 7 days, but there was no diminishing effect on root and shoot growth. Control (no TNT) plants were used to study the effect of TNT on growth. After 12 days, plant-, and urea-free soil pots treated with 40 and 80 mg kg1 initial TNT loads showed 27% and 7.5% decrease in TNT respectively (Fig. 1). After the completion of the phytoextraction experiment, no significant (p > 0.05) difference was observed for the soil TNT concentrations between 12 and 48 days. The small decrease in the soil-TNT concentrations observed in the absence of vetiver grass and urea could be ascribed to the indigenous microbial population. Vetiver grass significantly (p < 0.001) decreased soil TNT concentrations (both in presence and absence of urea) compared to
A 10 0
the TNT amended-no-plant controls (Fig. 2). After 3 days, TNT reduction by vetiver grass from soil treated with 40 mg kg1TNT reached 97% (Fig. 2A) and remained unchanged until the 12th day (Fig. 2B). Doubling the initial TNT concentration (80 mg kg1), resulted in 39% and 88% TNT removal by vetiver grass after 3 and 12 days, respectively (Fig. 2A, B). Pavlostathis et al. (1998) reported that TNT disappearance from soil was a function of both plant concentration and initial TNT concentrations. TNT removal by different plants in hydroponic media (Adamia et al., 2006; Makris et al., 2007b,c) as well as from soil (Scheidemann et al., 1998) decreased with increasing TNT concentrations. In accordance with our hydroponic results (Makris et al., 2007b), this soil-pot-experiment suggests gradual saturation of vetiver’s TNT adsorption capacity with increasing initial TNT loads. Addition of urea significantly (p < 0.001) enhanced the soil-TNT removal by vetiver grass. After 12 days, complete removal of TNT was observed in soils treated with 40 mg kg1 TNT. However, at this TNT load, no significant difference was observed in soil-TNT concentrations between the plant treatments with urea (100% TNT removal) or without (97% TNT removal), masking any urea effect. At 80 mg kg1 TNT load, soil-TNT concentration decreased by 84% in the presence of urea within 3 days, while in the absence of urea only 39% was removed by vetiver (Fig. 2). After 12 days, urea-vetiver system achieved 95% TNT removal, which was significantly higher than the untreated (no urea) vetiver treatment (84% removal). Pseudo first order (k1) and plant-normalized second order (kp) reaction rate constants were calculated to describe TNT removal kinetics by vetiver grass in the presence and absence of urea (Table 1). Results show, k1 and kp values were higher in urea treatments when compared to the untreated (no urea) controls. However, after 3 days, the differences in these rate constants between urea treated and untreated pots at the lower TNT treatments were not significant, suggesting that urea effect was masked by the high affinity of vetiver grass for TNT at lower initial load. Similar rate constants in higher TNT concentration after 12 days can be explained by the phytotoxic effects that were observed after 7 days in vetiver grass exposed to 80 mg kg1 TNT. k1 values at 40 mg kg1 TNT treatments were lower than those reported by Makris et al. (2007b) in the hydroponic systems with 40 mg l1 initial aqueous TNT concentrations. In the absence of urea, the k1 value obtained in the present soil study (k1 ¼ 0.014 h1) was significantly lower than the k1 (0.029 h1) reported in the hydroponic study. In hydroponic system, TNT was readily available to plants whereas soil-bound TNT was less available for plant uptake. In the presence of urea, these values were not significantly different from each other
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Fig. 1. Residual TNT in soils (mg kg1) initially treated with 40 mg kg1 (1A) and 80 mg kg1 (1B) TNT in plant-free, TNT-amended controls. Data are expressed as mean (n ¼ 3) 1 standard deviation.
P. Das et al. / Environmental Pollution 158 (2010) 1980–1983
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Fig. 2. Residual TNT in soils (mg kg1) initially treated with 40 mg kg1 and 80 mg kg1 TNT with two urea concentrations (0 and 1000 mg kg1) in presence of vetiver grass after 3 days (2A) and 12 days (2B). Data are expressed as mean (n ¼ 3) 1 standard deviation.
(k1soil ¼ 0.022 h1; k1hydroponic ¼ 0.026 h1). This dataset indicated that the presence of urea helped to release the soil-bound TNT to solution and hence enhanced its phytoavailibility. Chaotropic effects of urea catalyzed TNT removal by vetiver grass from soil due to the water structure modifications around soil particle surfaces that increased TNT solubility at particle/solution interface and thus enhanced potential for adsorption by root hair. This preliminary soil-pot-experiment validates the encouraging results obtained in the hydroponic studies (Makris et al., 2007a,b,c). The urea-stimulated phytoremediation method for a TNTcontaminated soil was effective in enhancing TNT phytoextraction from soil (Makris et al., 2010). The enhanced rate of phytoextraction of TNT in the urea treatment suggested that urea facilitated the release of soil-bound TNT into soil solution, making it more phytoavailable. However, the processes governing urea-catalyzed release of previously sorbed TNT from soil need to be investigated in both the presence/absence of plants. The 1000 mg kg1 urea that we have used in this study is the maximum urea that can be applied without affecting the soil health and environmental safety. In this preliminary study we have determined the chaotropic effectiveness of urea at its highest possible application rate in soil to enhance the TNT removal by vetiver grass from soil. Phytoremediation of TNTcontaminated soils that are not going to be used for agricultural purposes may afford up to 1000 mg kg1 urea application rates. Recommended urea application rates for agricultural crops (125– 350 mg kg1) were lower than that used in this study (EFMA, 2000; Fenn et al., 1987; Trierweiler and Omar, 1983). Additional greenhouse soil-pot-experiments are underway to evaluate the effect of Table 1 Reaction rate constants during TNT removal from soil using vetiver grass. Time (day)
Urea (mg kg1)
Initial TNT (mg kg1)
Pseudo first order rate constant k1(h1)
Plant-normalized second order rate constant kp (kg d1 g1)
3 3 3 3 12 12 12 12
0 1000 0 1000 0 1000 0 1000
40 40 80 80 40 40 80 80
0.051 0.01a 0.062 0.01a 0.007 0.00a 0.026 0.00b 0.014 0.00a 0.022 0.00b 0.007 0.00a 0.015 0.01a
0.041 0.01a 0.050 0.01a 0.006 0.00a 0.020 0.00b 0.011 0.00a 0.017 0.00b 0.006 0.00a 0.012 0.01a
Plant density was 30 g kg1. The kp values were calculated by dividing k1 by the plant density. Mean separation was conducted for each initial TNT concentrations for each day separately. Treatments with different superscript letters are significantly different at the 95% confidence interval.
urea as a chaotropic agent using agronomically recommended application rates. Further studies on the proposed stimulative phytoremediation method are necessary to ascertain the extent of TNT sequestration by vetiver grass as well as its transformation within the plant tissue.
Acknowledgement We thank Ms. Jamie Koshiba, Mr. Emeka Ovuegbe, and Ms. Gloria Adame for assisting with the experiments.
References Adamia, G., Ghoghoberidze, M., Graves, D., Khatisashvili, G., Kvesitadze, G., Lomidze, E., Ugrekhelidze, D., Zaalishvili, G., 2006. Absorption, distribution, and transformation of TNT in higher plants. Ecotox. Environ. Saf. 64 (2), 136–145. Eriksson, J., Skyllberg, U.J., 2001. Binding of 2,4,6-trinitrotoluene and its degradation products in a soil organic matter two-phase system. J. Environ. Qual. 30, 2053–2061. European Fertilizer Manufacturers Association (EFMA), 2000. Code of best agricultural practice: Urea. Available from http://www.efma.org. Fenn, L.B., Malstrom, H.L., Wu, E., 1987. Ammonia losses from surface applied urea as related to urea application rates, plant residue and calcium chloride addition. Nutr. Cycl. Agroecos 12, 219–227. Hughes, J.B., Shanks, J., Vanderford, M., Lauritzen, J., Bhadra, R., 1997. Transformation of TNT by aquatic plants and plant tissue cultures. Environ. Sci. Technol. 31, 266–271. Makris, K.C., Shakya, K.M., Datta, R., Sarkar, D., Pachanoor, D., 2007a. High uptake of 2,4,6-trinitrotoluene by vetiver grassdpotential for phytoremediation? Environ. Pollut. 146, 1–4. Makris, K.C., Shakya, K.M., Datta, R., Sarkar, D., Pachanoor, D., 2007b. Chemicallycatalyzed uptake of 2,4,6-trinitrotoluene by Vetiveria zizanioides. Environ. Pollut. 148, 101–106. Makris, K.C., Datta, R., Sarkar, D., Shakya, K.M., Pachanoor, D., Das, P., 2007c. Chaotropic effects on 2,4,6-trinitrotoluene uptake by wheat (Triticum aestivum). Plant Soil 295, 229–237. Makris, K.C., Sarkar, D., Datta, R., 2010. Coupling indigenous biostimulation and phytoremediation for the restoration of 2,4,6,-trinitrotoluene-contaminated sites. J. Environ. Monit., doi:10.1039/b908162c. Pavlostathis, S.G., Comstock, K.K., Jacobson, M.E., Saunders, F.M., 1998. Transformation of 2,4,6-trinitrotoluene by the aquatic plant Myriophyllum spicatum. Environ. Tox. Chem. 17, 2266–2273. Pennington, J.C., Patrick, W.H., 1990. Adsorption and desorption of 2,4,6- trinitrotoluene by soils. J. Environ. Qual. 19, 559–567. Sall, J., Creighton, L., Lehman, A., 2005. JMP Start Statistics, third ed. SAS Institute, Cary, NC/Pacific Grove, CA. Sarkar, D., Datta, R., Sharma, S., 2005. Fate and bioavailability of arsenic in organoarsenical pesticide-applied soils: part-I: incubation study. Chemosphere 60, 188–195. Scheidemann, P., Klunk, A., Sens, C., Werner, D., 1998. Species dependent uptake and tolerance of nitroaromatic compounds by higher plants. J. Plant Physiol. 152, 242–247.
P. Das et al. / Environmental Pollution 158 (2010) 1980–1983 Thompson, P.L., Ramer, L.A., Schnoor, J.L., 1998. Uptake and transformation of TNT by hybrid poplar trees. Environ. Sci. Technol. 32, 975–980. Trierweiler, J.F., Omar, M.F., 1983. Urea rate and placement for maize production on a calcareous vertisol. Nutrient Cycling in Agroecosystems 4, 261–270. U.S. EPA, 1991. Risk Assessment Guidance for Superfund vol. 1, Human OSWEF. Directive Publ. 25 March Health Evaluation Manual, Supplemental Guidance 9285, 6–03.
1983
U.S. EPA, 1997. Method 8330, Nitroaromatics and nitramines by high-performance liquid chromatography. Draft Update IVB SW-846, Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, USEPA-65 FR 70678, U.S. Governmental Printing Office: Washington, DC. Xiao, H., Zhou, Q.X., Liang, J.D., Jun–Sep 2004. Single and joint effects of acetochlor and urea on earthworm Esisenia foelide populations in phaiozem. Environ. Geochem. Health 26 (2–3), 277–283.
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Short communication
Vetiver grass is capable of removing TNT from soil in the presence of urea Padmini Das a, Rupali Datta b, Konstantinos C. Makris c, *, Dibyendu Sarkar a a
Department of Earth and Environmental Studies, Montclair State University, One Normal Avenue, Montclair, NJ 07104, USA Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA c Cyprus International Institute for Environmental and Public Health in Association with Harvard School Of Public Health, Cyprus University of Technology, Limassol, Cyprus b
Vetiver grass in the presence of urea effectively removes TNT from soil.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2009 Received in revised form 3 December 2009 Accepted 5 December 2009
The high affinity of vetiver grass for 2,4,6 trinitrotoluene (TNT) and the catalytic effectiveness of urea in enhancing plant uptake of TNT in hydroponic media we earlier demonstrated were further illustrated in this soil-pot-experiment. Complete removal of TNT in urea-treated soil was accomplished by vetiver at the low initial soil-TNT concentration (40 mg kg1), masking the effect of urea. Doubling the initial TNT concentration (80 mg kg1) significantly (p < 0.002) increased TNT removal by vetiver, in the presence of urea. Without vetiver grass, no significant (p ¼ 0.475) change in the soil-TNT concentrations was observed over a period of 48 days, suggesting that natural attenuation of soil TNT could not explain the documented TNT disappearance from soil. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: 2,4,6-trinitrotoluene (TNT) Urea Phytoremediation Chaotropic effect Bioremediation
1. Introduction The U.S. Environmental Protection Agency (U.S. EPA) has classified 2,4,6-trinitrotoluene (TNT) as a group C human carcinogen (U.S. EPA, 1991). Downward migration of TNT to groundwater from explosive-contaminated sites and related wastewater lagoons is of serious concern. Numerous military sites in the U.S. are in the process of being transferred to non-military entities under the base realignment and closure (BRAC) program. Following army base closures, military land may be offered to the public, but residual soil-TNT concentrations prohibit change of land use, unless appropriate remedial measures are taken. High costs and environmental concerns associated with most ex-situ remedial practices for TNT-contaminated soils have built interest in in-situ bioremediation practices (Makris et al., 2010). Our group has been investigating novel in-situ bioremediation methods for the restoration of TNT-contaminated sites. In a previous hydroponic study, we showed that vetiver grass exhibited high uptake capacity for soluble TNT (Makris et al., 2007a). The current study performed in a greenhouse setting showed that vetiver can remove TNT from soil as well, by utilizing the stimulative phytoremediation method (Makris et al., 2010). Stimulative phytoremediation is an in-situ bioremediation method for nitroaromatics that stems from the
synergistic combination of phytoremediation and biostimulation via the use of nutrient/chaotropic agent amendments (Makris et al., 2010). The limited phytoavailability of soil-TNT prompted us to test the stimulative phytoremediation method, using urea as a chaotropic agent, to enhance the solubility and plant uptake of TNT. Addition of urea altered the water structure, reducing the thermodynamic barrier associated with the introduction of a hydrophobic compound (TNT), thus increasing TNT solubility and plant uptake in a hydroponic setup (Makris et al., 2007a). Under conditions of similar initial TNT concentration, vetiver grass was superior to other plant species in removing TNT from aqueous media (Makris et al., 2007a,b), but its ability to take up TNT from soil is yet to be evaluated. Soil properties play an important role in controlling soil particle-bound TNT availability to plants/ trees and soil biological organisms (Pennington and Patrick, 1990). Eriksson and Skyllberg (2001) showed that mobility of TNT in soil primarily depended upon the soil organic matter (SOM) content. This short study was conducted to test the effectiveness of stimulative phytoremediation using the urea-vetiver system in enhancing TNT removal from Immokalee soil which is characterized by low SOM. The objective of this study was to evaluate the effectiveness of urea as a chaotropic agent in enhancing TNT removal by vetiver grass from TNT-contaminated soil. 2. Methods
* Corresponding author. Tel.: þ357 25002398. E-mail address: [email protected] (K.C. Makris). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.12.011
The soil-pot study was conducted in a greenhouse setting with the following treatments: i) three TNT concentrations (0, 40, 80 mg kg1); and ii) two chaotropic
P. Das et al. / Environmental Pollution 158 (2010) 1980–1983 agent (urea) concentrations (0 and 1000 mg urea kg1). Assuming that TNT would be less available for plant uptake from soil when compared to the hydroponic system (Thompson et al., 1998), 1000 mg urea kg1 (1045 kg urea-N ha1assuming a 15 cm soil depth) rate was used, which was the highest urea concentration tested during the hydroponic study (Makris et al., 2007b,c). This is also the highest concentration of applied urea that complies with current agronomic and environmental guidelines (Xiao et al., 2004). The Immokalee soil (pH 6, >90% sand, and 0.8% soil organic matter) (Sarkar et al., 2005) was collected from the surface horizon in the Southwest Florida Research and Education Center, Immokalee, Florida. Vetiver plants were allowed to acclimatize for 2 weeks in uncontaminated (no TNT) Immokalee soil. After two weeks, plants were transferred to the TNT-spiked soil pots, reaching a uniform plant density of 30 0.5 g kg1. Three TNT-free control soil pots were set up with vetiver grass to compare the potential toxic effects of TNT on TNT-amended plants. Six plant- and urea-free, TNTamended soil pots (40 and 80 mg kg1TNT) were also included to investigate any TNT losses due to biodegradation. All treatments were performed in triplicates. Pots were wrapped with aluminum foil to prevent potential TNT photodegradation. Experiments were carried out until near complete removal of TNT (12 days) from the spiked soil with 40 mg kg1 TNT. Soil samples were collected after 3 days to monitor soil TNT removal kinetics by vetiver grass. Soil microbial community can play major role in decreasing soil TNT by transforming TNT to metabolites (Hughes et al., 1997). The biological augmentation of TNT in soil was investigated by including plant-free, TNT-amended soil pots in the greenhouse for 48 days; soil samples were collected after 0, 12, 22, 32, 41, and 48 days for TNT estimation. Residual TNT in soil was extracted using the USEPA 8330 method, and analyzed using HPLC system (Prostar, Varian inc., USA) equipped with a UV/VIS absorbance detector (U.S. EPA, 1997; Makris et al., 2007b). Reaction rates of TNT removal by vetiver grass from soil were calculated as described by Pavlostathis et al. (1998), and Makris et al. (2007b). Statistical analyses were performed using the JMP IN version 5.1 (Sall et al., 2005).
3. Results and discussion After 12 days of exposure to soil TNT, vetiver plants did not show any phytotoxic symptoms for the 40 mg kg1 TNT load. For the 80 mg kg1 TNT load, vetiver developed yellow coloration on leaves after 7 days, but there was no diminishing effect on root and shoot growth. Control (no TNT) plants were used to study the effect of TNT on growth. After 12 days, plant-, and urea-free soil pots treated with 40 and 80 mg kg1 initial TNT loads showed 27% and 7.5% decrease in TNT respectively (Fig. 1). After the completion of the phytoextraction experiment, no significant (p > 0.05) difference was observed for the soil TNT concentrations between 12 and 48 days. The small decrease in the soil-TNT concentrations observed in the absence of vetiver grass and urea could be ascribed to the indigenous microbial population. Vetiver grass significantly (p < 0.001) decreased soil TNT concentrations (both in presence and absence of urea) compared to
A 10 0
the TNT amended-no-plant controls (Fig. 2). After 3 days, TNT reduction by vetiver grass from soil treated with 40 mg kg1TNT reached 97% (Fig. 2A) and remained unchanged until the 12th day (Fig. 2B). Doubling the initial TNT concentration (80 mg kg1), resulted in 39% and 88% TNT removal by vetiver grass after 3 and 12 days, respectively (Fig. 2A, B). Pavlostathis et al. (1998) reported that TNT disappearance from soil was a function of both plant concentration and initial TNT concentrations. TNT removal by different plants in hydroponic media (Adamia et al., 2006; Makris et al., 2007b,c) as well as from soil (Scheidemann et al., 1998) decreased with increasing TNT concentrations. In accordance with our hydroponic results (Makris et al., 2007b), this soil-pot-experiment suggests gradual saturation of vetiver’s TNT adsorption capacity with increasing initial TNT loads. Addition of urea significantly (p < 0.001) enhanced the soil-TNT removal by vetiver grass. After 12 days, complete removal of TNT was observed in soils treated with 40 mg kg1 TNT. However, at this TNT load, no significant difference was observed in soil-TNT concentrations between the plant treatments with urea (100% TNT removal) or without (97% TNT removal), masking any urea effect. At 80 mg kg1 TNT load, soil-TNT concentration decreased by 84% in the presence of urea within 3 days, while in the absence of urea only 39% was removed by vetiver (Fig. 2). After 12 days, urea-vetiver system achieved 95% TNT removal, which was significantly higher than the untreated (no urea) vetiver treatment (84% removal). Pseudo first order (k1) and plant-normalized second order (kp) reaction rate constants were calculated to describe TNT removal kinetics by vetiver grass in the presence and absence of urea (Table 1). Results show, k1 and kp values were higher in urea treatments when compared to the untreated (no urea) controls. However, after 3 days, the differences in these rate constants between urea treated and untreated pots at the lower TNT treatments were not significant, suggesting that urea effect was masked by the high affinity of vetiver grass for TNT at lower initial load. Similar rate constants in higher TNT concentration after 12 days can be explained by the phytotoxic effects that were observed after 7 days in vetiver grass exposed to 80 mg kg1 TNT. k1 values at 40 mg kg1 TNT treatments were lower than those reported by Makris et al. (2007b) in the hydroponic systems with 40 mg l1 initial aqueous TNT concentrations. In the absence of urea, the k1 value obtained in the present soil study (k1 ¼ 0.014 h1) was significantly lower than the k1 (0.029 h1) reported in the hydroponic study. In hydroponic system, TNT was readily available to plants whereas soil-bound TNT was less available for plant uptake. In the presence of urea, these values were not significantly different from each other
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Fig. 1. Residual TNT in soils (mg kg1) initially treated with 40 mg kg1 (1A) and 80 mg kg1 (1B) TNT in plant-free, TNT-amended controls. Data are expressed as mean (n ¼ 3) 1 standard deviation.
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80
Initial TNT load (mg kg -1)
Fig. 2. Residual TNT in soils (mg kg1) initially treated with 40 mg kg1 and 80 mg kg1 TNT with two urea concentrations (0 and 1000 mg kg1) in presence of vetiver grass after 3 days (2A) and 12 days (2B). Data are expressed as mean (n ¼ 3) 1 standard deviation.
(k1soil ¼ 0.022 h1; k1hydroponic ¼ 0.026 h1). This dataset indicated that the presence of urea helped to release the soil-bound TNT to solution and hence enhanced its phytoavailibility. Chaotropic effects of urea catalyzed TNT removal by vetiver grass from soil due to the water structure modifications around soil particle surfaces that increased TNT solubility at particle/solution interface and thus enhanced potential for adsorption by root hair. This preliminary soil-pot-experiment validates the encouraging results obtained in the hydroponic studies (Makris et al., 2007a,b,c). The urea-stimulated phytoremediation method for a TNTcontaminated soil was effective in enhancing TNT phytoextraction from soil (Makris et al., 2010). The enhanced rate of phytoextraction of TNT in the urea treatment suggested that urea facilitated the release of soil-bound TNT into soil solution, making it more phytoavailable. However, the processes governing urea-catalyzed release of previously sorbed TNT from soil need to be investigated in both the presence/absence of plants. The 1000 mg kg1 urea that we have used in this study is the maximum urea that can be applied without affecting the soil health and environmental safety. In this preliminary study we have determined the chaotropic effectiveness of urea at its highest possible application rate in soil to enhance the TNT removal by vetiver grass from soil. Phytoremediation of TNTcontaminated soils that are not going to be used for agricultural purposes may afford up to 1000 mg kg1 urea application rates. Recommended urea application rates for agricultural crops (125– 350 mg kg1) were lower than that used in this study (EFMA, 2000; Fenn et al., 1987; Trierweiler and Omar, 1983). Additional greenhouse soil-pot-experiments are underway to evaluate the effect of Table 1 Reaction rate constants during TNT removal from soil using vetiver grass. Time (day)
Urea (mg kg1)
Initial TNT (mg kg1)
Pseudo first order rate constant k1(h1)
Plant-normalized second order rate constant kp (kg d1 g1)
3 3 3 3 12 12 12 12
0 1000 0 1000 0 1000 0 1000
40 40 80 80 40 40 80 80
0.051 0.01a 0.062 0.01a 0.007 0.00a 0.026 0.00b 0.014 0.00a 0.022 0.00b 0.007 0.00a 0.015 0.01a
0.041 0.01a 0.050 0.01a 0.006 0.00a 0.020 0.00b 0.011 0.00a 0.017 0.00b 0.006 0.00a 0.012 0.01a
Plant density was 30 g kg1. The kp values were calculated by dividing k1 by the plant density. Mean separation was conducted for each initial TNT concentrations for each day separately. Treatments with different superscript letters are significantly different at the 95% confidence interval.
urea as a chaotropic agent using agronomically recommended application rates. Further studies on the proposed stimulative phytoremediation method are necessary to ascertain the extent of TNT sequestration by vetiver grass as well as its transformation within the plant tissue.
Acknowledgement We thank Ms. Jamie Koshiba, Mr. Emeka Ovuegbe, and Ms. Gloria Adame for assisting with the experiments.
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